Radiommunoassay determining the hepatitis associated antigen content of blood

ABSTRACT

Antibodies to serum hepatitis are coupled to carbohydrate macro particles which are then contacted with blood fluids, suitably plasma, which are believed to contain serum hepatitis antigen. The serum hepatitis antigen is thus complexed with the bound antibody and the particles bearing both the antibody and the antigen are removed from the blood fluid. The antigen is then cleaved from the antibody by alkaline treatment, and may, if desired, be itself isolated, suitably by dialysis. The antibody/agarose complex is then available for recycling in a further batch of contaminated blood fluids. There is also disclosed a novel method of highly sensitive measurement of the extent of contamination of blood fluids with serum hepatitis antigen.

United States Patent 11 1 Charm et al.

1 1 RADIOMMUNOASSAY DETERMINING THE HEPATITIS ASSOCIATED ANTIGEN CONTENTOF BLOOD [75] inventors: Stanley E. Charm. Newton; Bing Lou Wong,Somerville, both of Mass.

[73] Assignee: Research Corporation, New York,

[22] Filed: July 13, 1972 21 App1.No.:271,452

[52] US. Cl. 424/]; 23/230 B [51] Int. Cl A6lk 27/04 [58] Field ofSearch 424/1. [2; 23/230 B; 250/106 T [56] References Cited UNITEDSTATES PATENTS 3,555,143 1/1971 Axcn ct al. .1 424/1 3,645,852 2/1972Axen ct al. t. 424/1 OTHER PUBLICATIONS Aach et al., Proc. Nat, Acad,Sci USA, Vol. 68, No. 5, pp. 1056-1060, May 1971,

[ 1 July 22, 1975 Wide, Radioimmunoassay Methods, Editors Kirkham andHunter, (1970).

Salmon et al., The Journal of Immunology, Vol. 104, No. 3, March 1970,pp. 665672,

Primary Examiner-Benjamin R. Padgett Attorney, Agent, or FirmOmri M,Behr [57] ABSTRACT Antibodies to serum hepatitis are coupled tocarbohydrate macro particles which are then contacted with blood fluids,suitably plasma, which are believed to contain serum hepatitis antigen.The serum hepatitis antigen is thus complexed with the bound antibodyand the particles bearing both the antibody and the antigen are removedfrom the blood fluid. The antigen is then cleaved from the antibody byalkaline treatment, and may, if desired, be itself isolated, suitably bydialysis. The antibody/agarose complex is then available for recyclingin a further batch of contaminated blood fluids. There is also discloseda novel method of highly sensitive measurement of the extent ofcontamination of blood fluids with serum hepatitis antigen.

4 Claims, 4 Drawing Figures SHEET PATENTEDJUL 2 2 ms 9 E o. 9 o. m .21

n n u 4 M M m h Haifa JOKFZOU mum. WELPdEUI m0 PATENTEDJUL 22 ms BHAB(lml) 20mg ANTI BODY SALINE SALINE pH ll SALINE pH 7.2

SHEET FIG.

PLASMA WITH HAA MIXING 8, 3OHRS- FILTER NO. I

WHATMAN PLASMA HAA FREE BHAB- HAA (lml) WASH AND FILTER SALINE WITHTRACES OF PLASMA BHAB-HAA DISSOCIATION OF BHAB-HAA SALINE WITH HAA1(5Om1) Pmmmwm I915 3 8 96,218

FIG. 4

TEST CONTROL BHAB PLASMA BHAB SALINE INCUBATE INCUBATE CENTRIFUGECENTRIFUGE AND WASH AND WASH ADD ADD HAA 1 HAA WASH WASH CENTRIFUGECENTRIFUGE 0R FILTER 0R FILTER COUNT OF RADIO coum' OF RADIO ACTIVITY BACTIVITY B RADIOMMUNOASSAY DETERMINING THE I'IEPATITIS ASSOCIATEDANTIGEN CONTENT OF BLOOD The invention described herein was made in thecourse of work under a grant or award from the Department of Health,Education and Welfare.

FIELD OF THE INVENTION Purification of serum hepatitis contaminatedblood.

DESCRIPTION OF THE PRIOR ART It is well known that a small, but. from aclinical point of view. statistically excessive proportion of bloodavailable for transfusion purposes is contaminated by serum hepatitis.This proportion rises steeply in the case of blood from commercialrather than volunteer sources. Heretofore. the vast majority of effortsto isolate serum hepatitis contamination from the patient who willreceive the blood or portions thereof have been directed to detection ofthe presence of serum hepatitis antigen therein. Even if these testswere substantially more sensitive than any available at this time. apositive reading showing contamination would cause the abandonment. fortransfusion purposes. of the batch of blood tested. In view of theextreme shortage of blood this is of its very nature an unfortunatelywasteful proceeding. On the other hand. a negative reading is noinsurance that the blood is totally uncontaminated. In the utilizationof red blood cells it is known to wash these cells to remove the serumhepatitis antigen therefrom. this procedure has been found satisfactory.however, it is extremely expensive. This method cannot. of course. beutilized for the purification of plasma which is one of the principalfluids utilized in blood transfusions. While whole blood has a verylimited shelf life even under optimum conditions, plasma has a fairlysubstantial shelf life, thus, it tends to be in great demand when freshwhole blood is not available.

The coupling of an antibody with an antigen is a well knownimmunological procedure which has found in its various modifications.considerable use as a basis of diagnostic tests wherein the contact ofthe two particles, one of which may be attached to a carrier, gives riseto agglutination in the test device. The basic problem with extendingthis method to removal of particles from, say. plasma. is that antibodyper so could not be added to the plasma since this would leave antibodycontamination in the blood.

A second problem lies in the very costly nature of serum hepatitisantibody. It appears that serum hepatitis is a disease which issubstantially confined to man although there are reports that thedisease may be caused in certain primates such as chimpanzees. Thus, anyantibody which is produced must originate from serum hepatitis antigenof human origin. Hence. from a commercial point of view. a purificationmethod utilizing antigen would only be practical where the nature of themethod permitted very substantial re-use of the antibody after theantigen extracted by it from the blood fluid had been removed. In orderto be viable such a procedure would have to operate with a regenerationloss of less than 1%. preferably of the order of U.l7 regeneration lossper cycle.

The coupling of an antibody to a suitable carrier such as agarose isknown. The most recent work is reported by Cuatrecasas in Bio. Chem.Bio. Phys. Res. Com.. 38, 947 (i970) which cites other work in thisarea. Cuatrecasas work reports the coupling of insulin antibodies to aparticulate form of agarose known Sepharose 2B utilizing the complex toremove small amounts of insulin from body fluids and regenerating thesepharose/antibody complex by removal of the insulin. This work.however, shows a regeneration loss of l()% per cycle which is vastly inexcess ofa commercially viable procedure. Thus, the discovery of asuitable mode of cleaving the antigen/antibody complex in an efficientmanner is of prime importance.

The amount of serum hepatitis antigen particles present in most bloodfluids such as plasma varies widely. however, such fluids generallycontain between It) and I0 particles per ml. Many methods of detectingserum hepatitis are known. however, in view of the practical problemscaused by the presence of small amounts of serum hepatitis antigenparticles much research effort has been directed to detecting traceamounts of antigen. Heretofore, it has been possible to use thetechnique of irnmunoelectro-osmophoresis (IEOP) to bring the level ofsensitivity to about l0 to l0 particles per ml. It is generallyestimated that this level of detection would only give a positivereading for from about 25 to about 50% of blood contaminated with serumhepatitis. It should be noted moreover. that the IEOP method requiressophisticated equipment and sophisticated operation. Certain radioimmuneassays have been developed Hollinger. et al (J. Immunol. 107. I099)disclose a technique wherein a test sample was incubated for 6 hourswith serum hepatitis antibody to which was added serum hepatitis antigenof known ""I radio activity. Incubation was continued for a further 18hours. and immuno precipatating antibody was then added to the mixtureand the precipitate separated out 24 hours later. This method permitsthe detection of particles down to the level of about If) through 10'"particles per ml. However, it will be seen that the time factor. 2 days,renders the test somewhat questionable for routine testing of bloodsamples prior to use. And. furthermore, an increase in its sensitivitywould be highly desirable.

A further problem associated with the techniques of study of serumhepatitis is the difficulty of obtaining pure serum hepatitis antibody.The techniques of antibody production are well known, however, if theantibody producing system is infused with proteinaceous material ofdifferent immunological characteristics it may produce antibodiescorresponding to each of those immunological characteristics. Thus, amode of isolation of high purity antigen is clearly to be considered ahighly desirable goal.

SUMMARY OF THE INVENTION In the process of the present invention serumhepatitis antibody (goat) is coupled to agarose. suitably Sepharose 28.by the cyanogen bromide method (See Borath. ct al. Nature, 2l5. l49l(1967)). The thus bound antibody. (hereinafter BHAB) is then mixed witha blood fluid. suitably plasma. and agitated for up to 30 hours. Theplasma is separated from the BHABI HAA complex by filtration. Thecomplex is dissociated by washing with saline at pH 9-12, suitably10.5-1 l .5 and the saline wash containing the HAA concentrated bydialysis. The BHAB is then washed again and utilized for the removal ofHAA from a further batch of plasma. In a modification of this procedure,the BHAB may be utilized to test the presence of HAA down to a level ofIO to 10 particles per ml. In this procedure BHAB is incubated with thetest plasma at about 37 for about 3 hours and a similar sample similarlyincu bated in saline to serve as control. The BHAB is separated from thetest plasma suitably by filtration or centrifugation and incubated for 1hour with "I-labeled hepatitis antigen. The control BHAB is of coursesimilarly treated. The radio activity associated with the test andcontrol samples of BHAB is then measured and the ratio compared to aplot of a similar ratio against the number of antigen particles in atest sample set forth on a standard curve produced by serial dilutionmethods. In place of agarose, filter paper may be used as a support forthe test procedure.

The method of HAA removal as set forth hereinabove, may be extended tothe preparation of hepatitis free fibrinogen, a clotting factor of greatutility in the treatment of the hemophilia and similar conditions. Inthis procedure, plasma is treated with BHAB as hereinabove, and furthertreated with agarose to which antifibrinogen has been coupled by similarmeans. The fibrinogen is then cleaved from the agarose/antifibrinogencomplex and is available for use as a hepatitis-free product.

DESCRIPTION OF THE DRAWINGS FIG. I is a graphic plot of HAA particlesper ml. of standardized fluid on the linear X axis plotted against theratio of radio activity of a BHAB/HAA/labeled HAA sample to the radioactivity of a BHAB/labeled HAA sample plotted on a logarithmic Y axis.

FIG. 2 is a multiple plot of the number of HAA particles shown to remainin a fluid containing said particles after treatment by the removalmethod of the present invention and recovered, by standard radio tracermethods, IEOP and the radio immuno assay method of the present inventionplotted on a logarithmic Y axis against contact time of the removalagent with the HAA contaminated fluid, plotted on a linear X axis.

FIG. 3 is a flow diagram of the HAA removal method of the presentinvention.

FIG. 4 is a flow diagram of the HAA detection method of the presentmethod.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the process of the presentinvention serum hepati tis antibody (suitably but not critically goatantibody) is coupled to agarose particles, preferably Sepharose 28 by amodification of the cyanogen bromide method. While agarose has beenfound particularly suitable as a support phase (hereinafter BHAB-A) theinvention is not limited thereto. Physically stable supports containingcarbohydrate groups may be employed. For example, cellusosic materialssuch as cotton cloth have been found operative, and, indeed high gradefilter paper is the carrier of choice in the testing modification of theinvention. In this procedure cyanogen bromide is agitated with thecarrier, suitably agarose and the pH increases to between 10 to 12,suitably to about pH II, and maintained at this level for from about 5to about 20 minutes, suitably for from about 8 to about l0 min utes. Theaqueous supernate is removed, suitably by filtration and the suspensionwashed at reduced temperatures suitably between about 0C and 10C,preferably at about 4C at pH from about 7 to about 8.5, suitably about7.8. To this suspension of activated agarose is added antibody. Theamount of antibody utilized will of course depend upon the titer of theantibody used. However, it has been found suitable to utilizeapproximately I50 milligrams of antibody to l0 ml. of say sepharosewhere the hemagglutination titer of the antibody is l:l28,()00. Contactbetween the antibody and the activated carrier is maintained for about12 hours with very gentle stirring in which time more than of theantibody is bound to the sepharose. The antibody/carrier complexsuitably antibody/sepharose complex (hereinafter BHAB) is thenthoroughly washed with a slightly alkaline wash, suitably 0.1 M sodiumbicarbonate at pH 8, therafter with an acetate buffer at about pH 4 andfinally with a tris buffer at pH 7.4. Suitably this last wash contains atrace suitably about 0.2% of bovine serum albumin. All steps are carriedout in the temperarange of 0 to 10C, suitably at about 8C.

In the preferred modification of the method for ex ample. in the removalof serum hepatitis antigen (hereinafter HAA) the BHAB, suitably BHAB-A,in con tacted with the plasma and the mixture agitated. The agitation issuitably carried out by means of gentle shaking. The contacting betweenthe BHABA and the plasma containing the HAA may take place at anytemperature between about 0C and 45C. The tempera ture decided upon willbe as a result of a compromise between two competing factors. Clearly,the higher the temperature the more rapid the reaction between theBHAB-A and the HAA. On the other hand, side reac tions and deteriorationof the plasma is less likely to occur ar lower temperatures. Generallyspeaking, it has been found most satisfactory to operate in thetemperature range of between 4C and 10C, preferably around 8C. At thistemperature it has been found by the method discussed hereinbelow thatthe number of HAA particles are reduced by approximately every 2 hoursin a substantially straightline logarithm against time relationship.Thus, over a period of about 24 to 30 hours, presuming an average numberof about lO particles per ml, under the general conditions of theprocess 99999999999999? of the particles of HAA will have been removed.(See FIG. 2) The procedure is operated utilizing approximately I ml. ofBHABA, the BHAB-A having 20 mg/ml of antibody protein as sociated withit, per about 8 ml. of plasma.

It is recognized that this forward projection beyond the presentsensitivity of the test method makes certain assumptions. Nevertheless,these assumptions are considered to be valid as the same approach istaken in the destruction of Clostridium botulinum in standard foodprocessing wherein the assumption is made that the Cl.botulinum isreduced to a safe level under certain conditions of sterilization in asituation where the test method available to the art at the present timeis of sufficient sensitivity to prove this postulate. [See Fundamentalsof Food Engineering, 2nd Ed., p. 189, Charm, Ari Pub. Co., Westport,Connecticut (197i )1.

The BHAB-A/HAA complex is then separated from the blood fluid. such asthe plasma. either by centrifugation or filtration. Either method isentirely satisfactory. Especially preferred however, is the use of afilter of polypropylene cloth.

The BHAB-A/HAA complex is then dissociated. The conditions ofdissociation have been found to be critical where it is desired torecycle the BHAB-A thus produced. The BHAB-A/HAA complex is washed withalkaline saline, suitably made alkaline with aqueous ammonia. A pH rangeof pH 9 to pH 12 is operative. however. at pH 12 the sepharose supportswells and while the antibody can be regenerated and reused at this pHsuch a high pH value is undesirable for purposes of multiple recycling.At pH 10.5 the dissociation rate is rather slow while the mostsatisfactory dissociation rate, that is to say, rapid without swellingof the se pharose occurs at pH 1 1. Hence, it is most desirable to workbetween pH [0.5 and pH ll.

in the dissociation step. the BHAB-A is subjected to several washes withthe saline suitably about 4 washes are employed utilizing about volumesof saline for each volume of BHAB-A. the contact time in each instancebeing from about 15 minutes to 1 hour.

The saline containing the HAA is separated by filtration and the BHAB-Ais then washed, suitably with substantially neutral saline, for example,saline at about pH 7.2, the wash separated and the regenerated BHAB-Areutilized in treating further samples of, say, plasma in accordancewith the methods set forth hereinabove.

The aforementioned saline wash containing the HAA is then dialyzed,suitably in dialysis tubing surrounded by polyethylene glycol. Theconcentration of HAA is increased l0O-fold from the wash. The alkalinesaline is removed thru the dialysis membrane. The ph of the HAAconcentrate is about 7.0. The HAA tested by immunoelectrophoresis isfound to be free of the plasma proteins it was originally associatedwith. HAA in the concentrated condition may have l0 to l0 particles/ml.

Utilizing the basic principles set forth in the present invention thedetection of HAA particles down to a level of at least l0" particles perml. may be carried out. in this procedure BHAB is incubated with testplasma, at the same time, a similar sample is incubated with neutralsaline as a control.

While quantities are not critical, it has been found satisfactory toutilize BHAB-A containing about milligrams of antibody (this wouldcorrespond to approximately 1 ml. of BHAB prepared as hereinabove).

While the test is operative with BHAB-A it has been found more suitableto proceed using an antibody/filter paper complex (BHAB-P). BHAB-P isprepared in a manner similar to that utilized for BHAB-A. In thecoupling step it has been found suitable to utilize filter paper ofabout 2,-50 sq.mm. in area. Whatman No. 3 chromatographic paper(manufactured by W&R Balston l.td., Maidenstone, England) has been foundespecially suitable. While the ratio of paper to antibody is notcritical, it has been found satisfactory to suspend 70 mg. of antibodyin a l0 ml. solution of ().l M sodium bicarbonate containing about 200pieces of filter paper of the foregoing dimensions.

The incubation is suitably carried out at between about and suitablyabout 37C for from about l to about 4, preferably for about 3 hours. TheBHAB is then separated by filtration or contrifugation and washed,suitably twice, with neutral saline.

Prior to the assay, HAA is labeled with or similar radioisotopecompatable with and attachable to proteins by methods unknown in theart. Approximately l0 pl. of antigen (HAA), having a radio activitycount of from about 2,000 to about 8,000 preferably about 5.000counts/min. in 0.5 ml. of neutral saline are incubated with the reactedBHAB-A or BHAB-P of the pre vious step. Preferably the incubation iscarried on for from about 30 to about 40, suitably about 37 for fromabout 30 minutes to about 2 hours, suitably for about l hour, with slowstirring.

The BHAB-A or BHAB-P carrying HAA from the test plasma and the "'llabeled HAA upon it, is then separated by centrifugation or, preferably,filtration and washed, suitably three times, with neutral saline untilno further radio activity is noted in the wash. The radio activity ofthe control carrier is then read by means known to the art. This countis designated as B. The control BHAB-A (or BHAB-P) utilized in the firststage of the present process is similarly treated with labeled antigen(HAA) and washed and the radio activity associated with said control iscounted and designated as B,,.

The actual number of particles of HAA per unit volume in the test plasmamay be determined by, for example, by a method of direct calculation orby determination from a graph.

In both methods the experimental procedures to gen erate the requireddata is the same.

The number of particles of HAA per unit volume in a standard sample ("lI is determined by known methods, such as lEOP which are not assensitive as the method of the present invention. Serial dilutions ofthis test sample are then prepared using bovine serum albumin in neutralsaline to produce a series of test samples of known fivalue, some ofthese diluted standards will of course be beyond the sensitivity of the[EOF test.

In accordance with the foregoing procedures the B value is determinedfor each of these diluted standards and designated "B units of radioactivity. The value of B is of course the same for any sample taken froma given batch of BHAB.

The radio activity of ratio of the BHAB reacted with test sample andlabeled HAA and that reacted with control and labeled HAA, 8/8,, and8/3,, is calculated for the unknown and the diluted standardsrespectively. in the latter case the number of HAA particles,"ficorresponding to "BIB is known. Therefore the statement may be madewhere k is a constant. Similarly,

B/B,,=Rfi

where k is as above. Therefore therefore B a, a

Hence [Tmay be directly calculated for any given EXAMPLE 1 Preparationof BHAB-A Five ml. of decanted Sepharose 2B is mixed with 300 mg. ofCNBr per ml. of Sepharose 2B. The pH is increased to 11 with 4N NaOH andmaintained at this level by titration for 8 to 10 minutes. Thesuspension is washed with 0.1 M NaHCO,;. (4C) pH 7.8, and stirred gentlyat 4C with 150 mg. of serum hepatitis antibody (goat, Electronucleonics.1nc., Bethesda. Maryland) in 10 ml. of mixture (hemagglutination titer lto 128,000). Based on absorbancy (280 p.) in the wash, about 77% of theantibody is bound to the sepharose. The thus formed BHAB-A was washedtwice with 10 times volume of 0.1 M NaHCO;, pH 8, twice with 10 timesvolume 0.1 M acetate buffer, pH 4, and twice with 10 times volume of 0.1M tris buffer, Ph 7.4, containing 0.2% of bovine sermum albumin (allsteps at 4C).

EXAMPLE 11 Plasma Extraction/Purification Antibody bound to Sepharose 2B(BHAB-A), l ml. (decanted volume with mg. protein) is mixed with plasma(about 8 ml.) containing HAA (obtained from Massachusetts Red Cross) at89C for 26 hours. Mixing is carried out with gentle stirring or shaking.Plasma is separated from the BHAB-A/HAA complex by filtration throughWhatman No. 1 filter paper. Small sam ples are withdrawn at varioustimes and tested for HAA.

FIG. I shows a plot of the ratio of HAA particles in BHAB-A treated HAAcontaining plasma to the number of particles in the original sample ofplasma, indicating the present limits of detectability in the standardradio tracer, lEOP and modified radio immuno assay technique of thepresent invention.

EXAMPLE [[1 Regeneration of BHAB The BHAB-A/HAA complex is dissociatedby washing with ammoniacal saline (4 X 10 ml.) at pH 11 per ml. ofcomplex, at C for a total of 120 minutes. The BHAB-A is washed with pH7.2 saline and finally dis tilled water. After a final vacuum filtrationto remove water, the BHAB-A is ready for use again. All washes for BHABare sterilized before use to prevent the development of microorganisms.

The regeneration efficiency of this system was mea sured by reactingabout 8 ml. of HAA containing plasma with a batch of BHAB-A originallycontaining 20.17 mg. of serum hepatitis antibody at 37. The regeneratedmaterial was contacted with a fresh sample of plasma from the same bach,and the time required for a negative HAA reading noted using the IEOPtechnique. These results indicate an increase in time between the secondand third regenerations, probably due to mechanical loss, but no furtherincrease in time between the third and twentieth regenerations,indicating that after a Shakedown in the initial runs the BHAB system isstable under the operating conditions of the present invention.

Time for Negative IEOP (hours) No. of Times Test No. Regcncratcd a.Standard Curve in order to determine the level of HAA contamination in asample of plasma, the BHAB-P used in the test must be standardized. Asample of plasma containing HAA particles (H particles per unit volume)measured by known techniques (i.e. radio tracer or IEOP) is prepared andsubjected to serial dilution with saline (pH 7) containing bovine serumalbumin (0.2% relative to saline). "BIB is then determined by the radioimmuno assay methods set forth hereinabove for a given batch of BHAB-Pand B/B plotted against H 17. Preparation of BHAB for the Modified RadioImmuno Assay for HAA About a hundred pieces of Whatman No. 3chromatography paper (6mm 6mm) are mixed with 0.6 gm. CNBr whileadjusting the pH valve to 11.0 by manual titration of 4N NaOH. Thislevel is maintained for 8-10 minutes. The papers are washed with 0.1 MNaHCO;, (4C, pH 7.8) and mixed with mg. of serum hepatitis antibody(goat, ENl Maryland) suspended in 10 ml. of mixture. The mixture isstirred very gently at 4C overnight. The thus formed BHAB-P is washedtwice with ml. of 0.1 M NaHCO pH 8, twice with 100 ml. 0.1 M acetatebuffer, pH 4, and twice with 10 ml. of 0.1 M tris buffer. pH 7.4.containing 0.2% of bovine sermum albumin (all steps at 4C). c. OperatingProcedure 1. Each piece of BHAB-P is added to one-half ml. of testplasma; then incubated at 37C for 3 hours. At the same time anotherpiece of BHAB-P should be mixed with one-half ml. of saline as acontrol. The incubation for both continues for 3 hours. 2. The BHABmoieties are separated from the plasma and saline respectively byfiltration and each washed twice with saline (5 ml., 25C).

3. lOul. of a previously prepared batch of lhepatitis antigen (5,000cpm) are incubated with each moiety of BHAB from Step 2 for 1 hour withslow stirring.

4, The BHAB-P papers are each separated and washed with saline threetimes (5 ml. each time, 25C).

5. The radio activity associated with each BHAB-P piece incubated withthe test sample is counted. This count is designated as B.

6. Similarly the radio activity associated with the antibody incubatedwith the control is counted and is designated as B The value of 8/8,, iscalculated and its correspondence with the number of particles of HAAper unit value (7-1 is read off from the standard curve produced inSection (a) of this Example.

We claim:

I. A method of determining the HAA content of a fluid comprising anunknown quantity of HAA particles per unit value designated 77,comprising the steps of:

l. Coupling serum hepatitis antibody to a support phase comprisingcarbohydrate groups by the sequential steps of treating said supportphase with cyanogen bromide, raising the pH of the mixture to between pHl0 and pH l2, removing the supernatant liquid, washing said supportphase at from about ph 7 to about pH 8, adding said serum hepatitisantibody to the mixture, washing the mixture free of unreacted antibody,buffering the support phase/antibody complex to pH 7.8 to pH 8 to yielda complex designated BHAB,

ll. lncubating a predetermined portion of the thus produced supportphase/antibody complex with the fluid containing HAA,

Ill. Washing the thus produced support phase/am tibody/HAA complexdesignated BHAB/HAA,

lV. incubating said BHAB/HAA with radio active HAA,

V. Washing the thus produced BHAB/HAA/radio active HAA complex,

Vl. Measuring the radio activity of said BHAB/ HAA/radio active HAAcomplex to obtain a value designated 8 units of radio activity,

Vll. incubating a predetermined portion of the support phase of Step (I)designated as BHAB using the same quantity thereof as utilized in Step(II) under the same physical conditions of incubation using neutralsaline as the incubation medium,

Vlll. Washing the product of Step (VII),

lX. Incubating the product of Step (Vlll) with radio active HAA selectedfrom the sample used in Step (IV) for the same time under the sameconditions of temperature, pressure and pH.

X. Washing the product of Step (lX),

Xi. Measuring the radio activity of the product of Step (X) to obtain avalue designated 8., units of radio activity,

X". Repeating Steps (ll) (Vl) utilizing a fluid containing apredetermined quantity of HAA particles per unit value designated as Hto obtain a value designated B units of radio activity.

2. A process according to claim 1 comprising the additional step of:

determining H in accordance with the formula 3. A process according toclaim 1 additionally comprising repeating the procedure of Step (Xll)thereof utilizing a plurality of solutions containing predeterminedquantities of HAA particles per unit value to obtain thereby a series ofvalues for the quantity B corresponding to predetermined values of F 4.A process according to claim 2 additionally comprising the steps ofcalculating an an rage value for fT/B and determining the value of H inaccordance with the formula

1. A METHOD OF DETERMINING THE HAA CONTENT OF A FLUID COMPRISING ANUNKNOWN QUANTITY OF HAA PARTICLES PER UNIT VALUE DESIGNATED H,COMPRISING THE STEPS OF: I. COUPLING SERUM HEPATITIS ANTIBODY TO ASUPPORT PHASE COMPRISING CARBOHYDRATE GROUPS BY THE SEQUENTIAL STEPS OFTREATING SAID SUPPORT PHASE WITH CYANOGEN BROMIDE, RAISING THE PH OF THEMIXTURE TO BETWEEN PH 10 AND PH 12, REMOVING THHE SUPERNATANT LIQUID,WASHING SAID SUPPORT PHASE AT FROM ABOUT PH 7 TO ABOUT PH 8, ADDING SAIDSERUM HEPATITIS ANTIBODY TO THE MIXTURE, WASHING THE MIXTURE FREE OFUNREACTED ANTIBODY, BUFFERING THE SUPPOORT PHASE/ANTIBODY COMPLEX TO PH7.8 TO PH 8 TO YIELD A COMPLEX DESIGNATED BHAB, II. INCUBATING APEDETERMINED PORTION OF THE THUS PRODUCED SUPPORT PHASE/ANTIBODY COMPLEXWITH THE FLUID CONTAINING HAA, III. WASHING THE THUS PRODUCED SUPPORTPHASE/ANTIBODY/HAA COMPLEX DESINGATED BHAB/HAA, IV. INCUBATING SAIDBHAB/HAA, V. WASHING THHE THUS PRODUCED BHAB/HAA/RADIO ACTIVE HAACOMPLEX, VI. MEASURING THE RATIO ACTIVITY OF SAID BHAB/HAA/RADIO ACTIVEHAA COMPLEX TO OBTAIN A VALUE DESIGNATED B UNITS OF RADIO ACTIVITY, VII.INCUBATING A PREDETERMINED PORTION OF THE SUPPORT PHASE OF STEP (I)DESIGNATED AS BHAB USING THE SAME QUANTITY THEREOF AS UTILIZED IN STEP(II) UNDER THE SAME PHYSICAL CONDITIONS OF INCUBATION USING NEUTRALSALINE AS THE INCUBATION MEDIU, VIII. WASHING THE PRODUCT OF STEP (VII),XI. MEASURING THE RADIO ACTIVITY OF THE PRODUCT OF STEP (X) HAA SELECTEDFROM THE SAMPLE USED IN STEP (IV) FOR THE SAME TIME UNDER THE SAMECONDITIONS OF TEMPERATURE, PRESSURE AND PH, X. WASHING THE PRODUCT OFSTEP (IX), XI. MEASURING THE RADIO ACTIVITY OF THE PRODUCT OF STEP (X)TO OBTAIN A VALUE DESGNATED BO UNITS OF RADIO ACTIVITY, XII. REPEATINGSTEPS (II) - (VI) UTILIZING A FLUID CONTAINING A PREDETERMINED QUANTITYOF HAA PARTICLES PER UNIT VALUE DESIGNATED AS SH TO OBTAIN A VALUEDESIGNATED SB UNITS OF RADIO ACTIVITY.
 2. A process according to claim 1comprising the additional step of: determining H in accordance with theformula
 3. A process according to claim 1 additionally comprisingrepeating the procedure of Step (XII) thereof utilizing a plurality ofsolutions containinG predetermined quantities of HAA particles per unitvalue to obtain thereby a series of values for the quantity sBcorresponding to predetermined values of sH.
 4. A process according toclaim 2 additionally comprising the steps of calculating an averagevalue for sH/sB and determining the value of H in accordance with theformula